After retrovirus-mediated transfer of the pluripotency-associated transcription factors Oct4, Sox2, Klf4, and c-Myc (a) in differentiated somatic cells (such as fibroblasts, b), a signaling network specific for pluripotent stem cells is established within 10 to 20 days. Most of the reprogramming protocols use either 3 or all 4 “Yamanaka”-factors to induce pluripotency in somatic cells. We investigated, which miRNAs improve the efficacy of iPS cell generation and identified an undescribed miRNA family (130b, 301, 721) modulating the expression of Meox2 (Pfaff et al., 2011).
Improving hepatic differentiation protocol that is applicable for both, hESCs and hiPSCs, we evaluated a cytokine and small molecule based protocol for direct differentiation of hESC and hiPS cells into hepatic cells (Sgodda et al., 2013). Furthermore, modulation of the Wnt-pathway during formation of definitive endoderm and during hepatic specification revealed an expandable population of CD117 and CD184 double positive cells. We now aim to FACsort these cells for further propagation in a co-culture system with murine fibroblasts. Analyses of their hepatic differentiation capabilities will be performed by qRT-PCR for endodermal markers (AFP, SOX17, GATA4 and FOXA2) and more specific hepatic cell markers (hHEX, ALB, HNF4, Cyp1A1).
In our recent studies we established three murine iPSC-based models for metabolic liver disorders:
- the copper storage disorder Wilson’s disease (toxic milk mice)
- alpha1-antitrypsin deficiency (PiZ mice)
- tyrosinemia type 1 (fumarylacetoacetate-hydrolase-deficiency, FAH-/- mice)
Hepatic precursor cells could be derived from these disease-specific iPSCs applying an in vitro differentiation protocol and could be visualized and selected by a lentiviral albumin-GFP/Neo construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease (Eggenschwiler et al., 2011).
Aiming at a sustained knock-down of a disease causing gene in iPSCs and their differentiated derivatives, we investigated a novel approach for the expression of a therapeutic shRNA in disease-specific iPSC using third generation lentiviral vectors. With our novel strategy, we were able to achieve a significant reduction in the expression of PiZ hA1AT in transduced and differentiated disease specific iPSC from a murine model. Next, were able to reproduce these findings in patient-specific iPSC generated from an individual suffering from severe alpha-1-antitrypsin deficiency with liver disease.